One of the main objectives of plant genetic engineering is to develop transgenic plants with new characteristics and traits, which may include insect resistance, virus resistance, herbicide resistance, yield enhancement, stress tolerance, nutritional improvement, expression of industrially valuable proteins in economically profitable expression systems, like plants, etc. Many factors contribute to high level expression of genes which code for such desired characteristics, where ‘expression’ includes transcription, translation and post translational events. The abundance of any one transcript in a cell directly relates to transcriptional events, which in turn depends upon the strength of the promoter from which it is expressed. Thus, for the development of transgenic plants where high level of transgene expression is to be obtained, it becomes absolutely indispensable that the transgene be expressed from a strong promoter, the transcript is stable, it is translated efficiently and that the resultant protein is also stable in plant cell. Each of these steps synergistically contributes to enhancing the level of expression of the product of the transgene.
A promoter can be defined as a pool of cis-acting elements, which work in co-ordination with trans acting transcriptional factors to achieve expression of the gene attached to it. A promoter provides an efficient docking site for RNA polymerase and the related accessory proteins, which in turn contribute to the transcription of the gene situated operably therewith. Thus, as mentioned, promoters are highly specialised DNA sequences which govern the time and efficiency of transcription. A promoter is classified as a constitutive promoter when it is operable almost equally at all times in a given organism, for example, the CaMV 35S promoter. Other promoters are tissue specific or inducible. The strength of a promoter varies depending on the frequency of initiation of transcriptional events. Depending on strength, promoters can further be classified as strong or weak.
Different types of promoters are required in plant biotechnology, depending upon the target use. Constitutive high level expression promoters are most useful to develop transgenic plants for high level production of commercially required proteins. Such high level expression is also desirable in several situations for modifying metabolic pathways and for improving plants to withstand a variety of stress situations.
Previous reports mainly deal with the identification of natural promoter elements in genes and their improvement. These include, the identification of the CaMV 35S promoter by Odell et al., Nature 313: 810-812 (1985), who had shown the strength and constitutive nature of CaMV 35S promoter. Later, Jensen et al. Nature 321: 669-674 (1986), Jefferson et al., EMBO J., 6: 390-3907 (1987), and Sander et al., Nucleic Acids Research, 4: 1543-1558 (1987), showed measurable levels of reporter gene mRNA expressed from 35S CaMV promoter in extracts prepared from leaves, stems, roots and flowers of transgenic plants. The CaMV 35S promoter has been widely used by scientists in the field of plant genetic engineering. Morelli et al., Nature (1985) 315:200-204 described that the CaMV 35S promoter is transcribed at a relatively high rate as evidenced by a ten-fold increase in transcription products as compared to the NOS promoter. Abel et al., Science (1986) 232:738-743, Bevan et al., EMBO J. (1985) 4: 1921-1926, Morelli et al., Nature (1985) 315:200-204, and Shah et al., Science (1986) 233:478-481 described that the 35S CaMV promoter is moderately strong and constitutively active. Therefore, the CaMV 35S promoter has been used to express a number of foreign genes in transgenic plants. Odell et al., Nature 313: 810-812 (1985), described that initiation of transcription from the 35S promoter is dependent on proximal sequences, which included a TATA element, while the rate of transcription was determined by sequences that were dispersed over 300 bp of upstream DNA. Simpson et al., Nature 323:551-554 (1986) described this region as an enhancer region (sequences which activate transcription are termed enhancers).
Subsequently, other workers tried to improve the CaMV promoter. Kay et al, Science 236: 1299-1302 (1987) duplicated a large region (253 bp) of the naturally existing CaMV 35S promoter and reported enhancement in its activity. Odell, et al., Plant Mol. Biol. 10:263-272 (1988), reported the use of a part of the CaMV 35S promoter as an enhancer in the nopaline synthase promoter. Mitsuhara, et al., Plant Cell Physiol. 37 (1): 49-59 (1996) compared many combinations of different CaMV 35S promoter sequence elements. By increasing the number of repeats of the native enhancer element, they obtained enhanced expression of the reporter gene. Ni, et al., The Plant Journal 7(4):661-676 (1995) combined portions of the naturally occurring octopine and mannopine synthase promoters to develop an efficient chimeric promoter. Ellis, et al., EMBO 6:11-16 (1987), reported the use of a natural octopine synthase promoter fragment to enhance the activity of the maize (adh-1) gene.
Other developments include identification of other natural promoter elements for expression of genes in plants. These include the use of the Figwort Mosaic Virus promoter for achieving enhanced expression per U.S. Pat. No. 5,378,619, Rubisco promoter as per U.S. Pat. No. 4,962,028, chimeric CaMV enhanced mannopine synthase promoter as per U.S. Pat. No. 5,106,739, enhanced CaMV 35S promoter as per U.S. Pat. No. 5,322,938, and the glutamine synthetase promoter for organ specific expression in plants as per U.S. Pat. No. 5,391,725.
As of now, attempts have been made to identify the naturally existing promoter sequences to be used as such or to exchange or rearrange parts of natural promoters so as to achieve a higher level of expression. However, in no case an attempt has been made to design an artificial promoter based on knowledge gained from computational analysis of various DNA sequences present upstream of the gene sequence, reported in the database.